Intercellular and/or intracellular signalling via receptor mediated activation of biochemical and/or molecular mechanisms is a fundamental process for regulating cellular and/or tissue homeostasis. Typically, ligands which interact with receptors to bring about a suitable biochemical response are known as agonists and those that prevent, or hinder, a biochemical response are known as antagonists. For example, and not by way of limitation, cell specific growth factors are ligands that act as agonists and bind receptors located at the cell surface. Activation of the receptors by ligand-specific binding promotes cell proliferation via activation of intracellular signalling cascades that result in the expression of, amongst other things, cell-cycle specific genes, and the activation of quiescent cells to proliferate. Growth factors may also activate cellular differentiation.
A large group of growth factors, referred to as cytokines, are involved in a number of diverse cellular functions. These include, by example and not by way of limitation, modulation of the immune system, regulation of energy metabolism and control of growth and development. Cytokines which are secreted by lymphocytes are termed lymphokines (also known as interleukins). Those secreted by monocytes and macrophages are termed monokines. Cytokines are also secreted by endocrine glands, (for example growth hormone (GH) by the pituitary gland), and adipose cells (for example leptin). Cytokines mediate their effects via receptors expressed at the cell surface on target cells.
Receptors of the cytokine receptor family possess a single transmembrane domain and lack intrinsic enzyme activity (1). Upon the binding of a cytokine to a cognate receptor, either receptor homo- or hetero-dimerisation or oligomerisation occurs. The receptor complex is internalised and signalling occurs through the activation of associated signalling cascades that include the Jak/Stat and Mapk pathways. Internalisation is followed by a recycling step whereby the receptor molecule is regenerated for further use within the cell.
The study of receptor/ligand interactions has been facilitated by the ability to define the structures of receptor molecules and their ligands. Several approaches, including X-ray crystallography and computer modelling, have greatly facilitated our understanding of the biology of ligand: receptor binding.
An example of the above is described with respect to GH and its binding to the growth hormone receptor (GHR). This example is merely meant to be illustrative and not limiting and is an example of a cytokine which activates a signal transduction cascade by binding, dimerisation and internalisation of the receptor:ligand complex.
It is known that a single molecule of growth hormone (GM) associates with two receptor molecules (3-6). This occurs through two unique receptor-binding sites on GH and a common binding pocket on the extracellular domain of two receptors. Site 1 on the GH molecule has a higher affinity than site 2, and receptor dimerization is thought to occur sequentially with one receptor binding to site 1 on GH followed by recruitment of a second receptor to site 2.
The extracellular domain of the GHR exists as two linked domains each of approximately 100 amino acids (SD-100), the C-terminal SD-100 domain being closest to the cell surface and the N-terminal SD-100 domain being furthest away. It is a conformational change in these two domains that occurs on hormone binding with the formation of the trimeric complex GHR-GH-GHR (FIG. 5). It has been proposed that ligand-driven receptor dimerization is the key event leading to signal activation (3), triggering phosphorylation cascades that include the Jak2/Stat5 pathway (7). Using confocal microscopy and Frequency Resonance Energy Transfer (FRET) it is known that there is very rapid internalisation of GHR after ligand binding and that internalisation and signalling are independent functions (16). Internalisation of the GHR-GH-GHR complex is followed by a recycling step whereby the receptor molecule is regenerated for further use within the cell.
The importance of receptor dimerization in signal transduction is indicated by a number of experiments. High concentrations of GH, which favour the monomeric GH-GHR complex, inhibit the GH signal (8). Mutations in the inter-receptor dimerization domain inhibit signalling without influencing GH binding (10). The strongest evidence comes from work with a GH molecule mutated at site 2 to prevent receptor dimerisation. These GH mutants block GH-stimulated cell proliferation (8, 11-14), the conformational change associated with receptor dimerization (15), and Jak-Stat signalling (16).
U.S. Pat. No. 5,849,535 describes a human growth hormone including a number of amino acid substitutions which disrupt Site 2 binding. The substitution of a different amino acid at G120 is one modification that disrupts Site 2 binding and the hGH variant acts as an hGH antagonist.
The in vivo efficacy of hGH and hGH variants is determined, in part, by their affinity for the hGH receptor and rate of clearance from the circulation. The kidneys are relatively small organs which receive approximately 25% of cardiac output. The kidneys perform several important functions primarily related to the regulation of the composition and volume of body fluids. The kidneys filter about 100 litres of plasma every day and of the blood flow in and out of a kidney only approximately 1% becomes urine. Approximately 20% of the plasma that passes through the kidney gets filtered into the nephron. Filtration takes place in the glomerulus which is driven by the hydrostatic pressure of the blood. Water and small molecules are filtered whereas blood cells and large molecules, for example polypeptides, do not pass through the glomerular filter.
Those polypeptides with an effective molecular weight above 70 kDa are not cleared by glomerular filtration because they are simply too large to be filtered. Certain proteins of small molecular weight are filtered by the glomerulus and are found in the urine. For example, Growth Hormone (GH has a molecular weight of 22.1 kDa and the kidney is responsible for clearing up to 60-70% of GH in humans (Baumann, 1991; Haffner et al, 1994), and up to 67% in rat (Johnson & Maack, 1977). Other examples of relatively small molecular weight polypeptides which are filtered by the kidney include lepin, erythropoeitin, and IL-6.
Syed et al (1997) constructed an anti-coagulant fusion protein which fused hirudin with albumin. This fusion protein showed extended plasma half life whilst maintaining a potent anti-thrombin (anti-coagulant) activity. This is likely to result from decrease in glomerular filtration by the kidneys. However a problem associated with this strategy is that hirudin is a foreign protein and which is known to provoke a strong immune response. The increase in molecular weight of the hirudin fusion protein increases the catabolic half-life from 0.7 hours to 4.6 days.
A further method to increase the effective molecular weight of proteins and to produce a product which has reduced immunogenicity is to coat the protein in polyethylene glycol (EG). The in-vivo half-life of GH has been increased by conjugating the proteins with poly ethylene glycol, which is termed “pegylation” (See Abuchowski et al., J. Biol Chem., 252:3582-3586 (1977). PEG is typically characterised as a non-immunogenic uncharged polymer with three water molecules per ethylene oxide monomer. PEG is believed to slow renal clearance by providing increased hydrodynamic volume in pegylated proteins (Maxfield et al., Polymer, 16:505-509 (1975)). U.S. Pat. No. 5,849,535 also describes humanGH (hGH) variants which are conjugated to one or more polyols, such as poly(ethylene glycol) (PEG).
An alternative to pegylation which provides a molecule which retains biological activity and is immune silent is herein disclosed. A chimeric protein comprising the extracelluar domain, or part thereof, of a receptor linked, via a variable flexible linker molecule to its cognate ligand to produce an agent with an apparent molecular weight greater than the native ligand. In the example provided, GH is fused to at least part of the growth hormone receptor (GHR) which gives an approximate molecular weight of 55 kDa which when glycosylated increases the effective molecular weight to approximately 75 kDa This would be of sufficient size to prevent the chimera being filtered by the kidney and, importantly, the molecule retains biological activity.
A long-acting form of growth hormone could be used in the treatment of both childhood and adult onset growth hormone deficiency. Growth hormone has well known anabolic actions and a long-acting form of growth hormone could be used for the treatment of a number of conditions by virtue of its anabolic actions including promoting growth in Turner's syndrome, renal failure, osteoporosis and muscle wasting in catabolic patients. Bovine somatotropin is currently used to enhance milk production from cows. A long-acting form of growth hormone would be an effective treatment for increasing milk yield from cows (Peel et al. 1981).
This strategy is applicable to other ligand-receptor combinations (eg. leptin, erythropoeitin and IL-6). For example, leptin is being trialed as a therapy for obesity (Mantzoros & Flier, 2000). A long-acting form of leptin could be used to treat obesity, insulin resistance, hyperlipidaemia and hypertension. Erythropoeitin is important in the generation of red cell mass. A long-acting form of erythropoeitin could be used to treat anaemia especially that associated with renal failure.
Truncated GH receptors, which lack the cytoplasmic domain of the receptor, act as dominant negative inhibitors of GH signalling (9,19). The truncated receptor is expressed at a high level on the cell surface because it lacks the cytoplasmic domain essential for internalisation (16). In the presence of GH, the truncated receptor heterodimerises with the fill length receptor and blocks signalling because it lacks the cytoplasmic domain. As the truncated receptor fails to internalise it acts as a dominant negative inhibitor preventing internalisation of the GH receptor complex.
The disorders of acromegaly and gigantism result from an excess of growth hormone, usually due to pituitary tumours. A drug currently under trial is the pegylated GH antagonist B2036, designed using recently acquired knowledge of the molecular structure of the growth hormone receptor (GHR). Unfortunately, high levels of B2036 are required to antagonise GH action with drug levels over a 1000 times higher than endogenous GH levels (18).
B2036, despite having a mutated site 2, binds to a receptor dimer, and is internalised in an identical fashion to GH. It does not however trigger the conformational change required for signalling. The high dose requirement of the antagonist relates to its internalisation and its differential binding to soluble and membrane bound receptor. The pegylated antagonist does not bind efficiently to membrane bound receptor although pegylation increases half-life and lowers immunogenicity. The non-pegylated antagonist is rapidly internalised and cleared.
There is a need to provide an antagonist that is not internalised by the cell and that can be delivered in lower doses. This would prove a more effective and potent antagonist and provide a more effective and economical treatment.
The leptin receptor (28) and erythropoietin (EPO) receptor (29,30) share considerable structural homology to the GHR and require a similar dimerisation process to trigger signalling. Leptin supresses appetite and leptin resistance is associated with obesity. A leptin receptor antagonist will provide a treatment for anorexia nervosa. EPO excess causes polycythaemia which may be secondary to hypoxia (chronic lung disease), or primary in the case of polycythaemia rubra vera (a disorder of excess red blood cells). An EPO antagonist will provide a therapy for polycythaemia.
A further example of a receptor: ligand binding is provided by the IL-6 activation of its cognate receptor. The current model for IL-6 activation of its cognate receptor stipulates that IL-6 binds to either soluble or membrane bound IL-6 receptor (IL-6R). The IL-6/UL-6R complex then recruits two molecules of gp130 and the tetramer signals through the dimerisation of the two gp130 molecules which possess cytoplasmic domains that associate with signalling molecules (Grotzinger et al., 1999). In nature UL-6 and the IL-6R exist as separate molecules which possess high affinity binding sites for each other and the association with the signal transducing molecule gp130 occurs through covalent linkage and the formation of disulfide bonds.
We have been studying receptor trafficking and binding protein production for two members of the cytokine receptor family; GH and leptin (9,16,20,21). These two hormones play a fundamental role in determining body composition in adults. Both leptin and GH are important in regulating energy expenditure, appetite, and fat mass. The ability to manipulate the biological actions of leptin and GH will have important therapeutic outcomes for the treatment of both hormone excess and deficiency.
Using confocal microscopy and Frequency Resonance Energy Transfer (FRET) we have shown that there is very rapid internalisation of GH receptor after ligand binding and that internalisation and signalling are independent functions (16). Our recent work shows that the GH antagonist, pegvisomant, despite having a mutated site 2, binds to a receptor dimer, is internalised in an identical fashion to GH, but does not trigger the conformational change required for signalling. We have demonstrated that the high dose requirement of the antagonist relates to its internalisation and its differential binding to soluble and membrane bound receptor. The pegylated antagonist does not bind efficiently to membrane bound receptor and the non-pegylated antagonist is rapidly internalised and cleared.
We demonstrate that a truncated GHE, which lacks the cytoplasmic domain of the receptor, can act as a dominant negative antagonist of GH signalling, (FIG. 5) (9,20). The truncated receptor is expressed at a high level on the cell surface as it lacks cytoplasmic domain essential for internalisation (16). The truncated receptor heterodimerises with the full length receptor, blocks signalling as it lacks the cytoplasmic domain, and acts as a dominant negative because it is present in excess on the cell surface and prevents internalization of the GH receptor complex.
There are two problems associated with using truncated receptors in the generation of antagonists to GH. A truncated receptor in the membrane would have to be generated from within the cell. The GHR is also proteolytically cleaved and in time the majority of the truncated receptor would be lost into the circulation.
We link GH, through its C-terminus and a linker, to the N-terminus of the C-terminal SD-100 domain of the GHR. By varying the length of the linker we define a molecule that has the flexibility to allow binding of GH through site 1 to full length receptor at the cell surface. The C-terminal SD-100 domain of the receptor will then rotate in to complete the trimeric structure GHR-GH-GHRtr where GHRtr is the C-terminal SD-100 domain. This complex neither signals nor internalises, and effectively antagonises GH action. It has the additional advantages of low immunogenicity and low clearance as the majority of GH is cleared via the GHR (22).
We also demonstrate that the leptin receptor produces a soluble binding protein (21) as do many cytokine receptors (2), and the predominant peripheral form of the leptin receptor is a truncated receptor similar to the truncated GHR (27,28). Our recent work has demonstrated that truncated leptin receptors can inhibit leptin signalling. The erythropoietin (EPO) receptor shares a very similar crystal structure to GHR and an EPO chimera with the C-terminal SD100 of the EPO receptor would function as an antagonist.